Nuclear localization of Chfr is crucial for its checkpoint function

School of Biological Sciences, Research Center for Functional Cellulomics, Seoul National University, Seoul 151-742, Korea.
Moleculer Cells (Impact Factor: 2.09). 04/2009; 27(3):359-63. DOI: 10.1007/s10059-009-0046-7
Source: PubMed


Chfr, a checkpoint with FHA and RING finger domains, plays an important role in cell cycle progression and tumor suppression. Chfr possesses the E3 ubiquitin ligase activity and stimulates the formation of polyubiquitin chains by Ub-conjugating enzymes, and induces the proteasome-dependent degradation of a number of cellular proteins, including Plk1 and Aurora A. While Chfr is a nuclear protein that functions within the cell nucleus, how Chfr is localized in the nucleus has not been clearly demonstrated. Here, we show that nuclear localization of Chfr is mediated by nuclear localization signal (NLS) sequences. To reveal the signal sequences responsible for nuclear localization, a short lysine-rich stretch (KKK) at amino acid residues 257-259 was replaced with alanine, which completely abolished nuclear localization. Moreover, we show that nuclear localization of Chfr is essential for its checkpoint function but not for its stability. Thus, our results suggest that NLS-mediated nuclear localization of Chfr leads to its accumulation within the nucleus, which may be important in the regulation of Chfr activation and Chfr-mediated cellular processes, including cell cycle progression and tumor suppression.

5 Reads
  • Source
    • "However, in hTERT-RPE1 cells CHFR was detected at centrosomes in growth-arrested as well as cycling cells (data not shown), suggesting that the lack of detection of CHFR at centrosomes of mitotic OSE cells could be due to low abundance of the protein. These data conflict with previous studies showing that over-expressed, epitope-tagged CHFR displays a predominantly nuclear localization [78-80], but are in agreement with studies showing that endogenous CHFR localizes to cytoplasm and centrosomes during interphase growth [31,81,82] and to spindle poles during mitosis [82]. This is the first report on CHFR localization to primary cilia, and future studies might reveal if CHFR takes part in the signaling machinery that regulates ciliary disassembly. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Ovarian cancer is the fourth leading cause of cancer-related deaths among women in Denmark, largely due to the advanced stage at diagnosis in most patients. Approximately 90% of ovarian cancers originate from the single-layered ovarian surface epithelium (OSE). Defects in the primary cilium, a solitary sensory organelle in most cells types including OSE, were recently implicated in tumorigenesis, mainly due to deregulation of ciliary signaling pathways such as Hedgehog (Hh) signaling. However, a possible link between primary cilia and epithelial ovarian cancer has not previously been investigated. The presence of primary cilia was analyzed in sections of fixed human ovarian tissue as well as in cultures of normal human ovarian surface epithelium (OSE) cells and two human OSE-derived cancer cell lines. We also used immunofluorescence microscopy, western blotting, RT-PCR and siRNA to investigate ciliary signaling pathways in these cells. We show that ovarian cancer cells display significantly reduced numbers of primary cilia. The reduction in ciliation frequency in these cells was not due to a failure to enter growth arrest, and correlated with persistent centrosomal localization of aurora A kinase (AURA). Further, we demonstrate that ovarian cancer cells have deregulated Hh signaling and platelet-derived growth factor receptor alpha (PDGFRα) expression and that promotion of ciliary formation/stability by AURA siRNA depletion decreases Hh signaling in ovarian cancer cells. Lastly, we show that the tumor suppressor protein and negative regulator of AURA, checkpoint with forkhead-associated and ring finger domains (CHFR), localizes to the centrosome/primary cilium axis. Our results suggest that primary cilia play a role in maintaining OSE homeostasis and that the low frequency of primary cilia in cancer OSE cells may result in part from over-expression of AURA, leading to aberrant Hh signaling and ovarian tumorigenesis.
    Cilia 08/2012; 1(1):15. DOI:10.1186/2046-2530-1-15
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Maintenance of genomic stability is needed for cells to survive many rounds of division throughout their lifetime. Key to the proper inheritance of intact genome is the tight temporal and spatial coordination of cell cycle events. Moreover, checkpoints are present that function to monitor the proper execution of cell cycle processes. For instance, the DNA damage and spindle assembly checkpoints ensure genomic integrity by delaying cell cycle progression in the presence of DNA or spindle damage, respectively. A checkpoint that has recently been gaining attention is the antephase checkpoint that acts to prevent cells from entering mitosis in response to a range of stress agents. We review here what is known about the pathway that monitors the status of the cells at the brink of entry into mitosis when cells are exposed to insults that threaten the proper inheritance of chromosomes. We highlight issues which are unresolved in terms of our understanding of the antephase checkpoint and provide some perspectives on what lies ahead in the understanding of how the checkpoint functions.
    Molecular and Cellular Biology 10/2009; 30(1):22-32. DOI:10.1128/MCB.00687-09 · 4.78 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: CHFR functions as a mitotic checkpoint by delaying entry into metaphase in response to mitotic stress. CHFR is frequently silenced by hypermethylation in human cancers, indicating that CHFR is a tumor suppressor. To further elucidate the role of CHFR in tumorigenesis, we studied the relationship between CHFR and a novel CHFR-interacting protein, HLTF, helicase-like transcription factor. Here we show that CHFR binds to and ubiquitinates HLTF, leading to its degradation. HLTF modulates basal expression of PAI-1 involved in regulation of cell migration. Consistently, overexpression of CHFR inhibits cell migration, resulting from reduced HLTF followed by decreased PAI-1 expression. HLTF expression is also higher in human breast cancer cells where CHFR is not expressed. Taken together, this is the first report identifying the regulatory mechanism of HLTF by CHFR, suggesting that CHFR-mediated downregulation of HLTF may help protect against cancer.
    Biochemical and Biophysical Research Communications 04/2010; 395(4):515-20. DOI:10.1016/j.bbrc.2010.04.052 · 2.30 Impact Factor
Show more